Genetička transformacija kičice (Centaurium erythraea Rafn.) AtCKX1 i AtCKX2 genima
Genetic transformation of Centaury (Centaurium erythraea Rafn.) using AtCKX1 and AtCKX2 genes
2012
Authors:
Trifunović-Momčilov, MilanaContributors
Dragićević, IvanaSubotić, Angelina
Simonović, Ana
Document Type:
Doctoral thesis (Published version)
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Show full item recordAbstract:
Plant species Centaurium erythraea Rafn., commonly known as common centaury, has
been used for centuries to cure febrile conditions, regulate blood sugar, treat anemia,
jaundice and gout and to increase appetite and stimulate digestion. Because of its numerous
therapeutic properties centaurii herba is officially recognized as a drug in a number of
pharmacopoeias. Root tips of ≈10 mm average length, isolated from seedlings obtained
under sterile conditions, were used as initial explants in this work. The in vitro culture of
non-transformed centaury roots was established on solid half-strength MS nutrient medium
without growth regulators. Organogenesis is a spontaneous process in centaury root culture.
The first changes were noticeable on the basal part of the initial explants, after seven days
of growth on MS medium. The root explants thickened and began to burst over the entire
length of the basal part. During four weeks of subculture, a large number of adventitious
buds were observed on initial explants. In order to obtain transgenic centaury plants with
lowered endogenous cytokinin levels, in vitro cultivated root explants were transformed
with Agrobacterium tumefaciens GV3101 bearing a pBinHTX plasmid with either AtCKX1
or AtCKX2 genes encoding isoforms of cytokinin oxidase/dehydrogenase (CKX). PCR
analysis of genomic DNA confirmed the effecient integration of both AtCKX1 (30%) and
AtCKX2 (28.2%) genes. In order to investigate the expression of AtCKX genes in transgenic
centaury plants, an optimization of PCR protocol with qPCR primers was necessary.
Optimization of PCR protocol implicated finding the optimal annealing temperature and
simultaneous application of different PCR additives and enhancers. Addition of 5% DMSO
significantly improved the specificity of qPCR amplification of AtCKX1 and AtCKX2
genes. qRT-PCR analysis indicated that AtCKX1 and AtCKX2 genes were expressed in 50%
and 64% of the analyzed transgenic lines, respectively. In this work we clearly demonstrate
a significant effect of AtCKX genes on cytokinin metabolism in transgenic centaury plants.
Since CKX is the only known enzyme involved in cytokinin catabolism, it is considered a
key factor in controlling the cytokinin levels in plant tissues. CKX enzyme activity was
determined in shoots and in roots of all AtCKX transgenic lines. The analysis of the content
of total cytokinins and individual groups of endogenous cytokinins in non-transformed
centaury plants enabled, for the first time, a determination of cytokinin profile of this
species in vitro. In this work we also showed that changes in the cytokinin metabolism have
unpredictable effects on the endogenous IAA levels in plant tissues. The expression of
AtCKX genes also affected some morphological traits of the transgenic centaury plants. We
observed that a decrease of endogenous cytokinins caused a reduction of morphogenetic
potential of transgenic centaury plants but did not significantly affect the biomass
production in comparison to control plants. Analysis of photosynthetic pigments of
centaury indicated that a decrease of endogenous cytokinins might be a prerequisite rather
than a signal for the onset of senescence. The main active pharmacological compounds of
the centaury are secoiridoids and xanthones. The content of the secoiridoids found in plants
grown in vitro was similar to that in plants collected from nature, whereas the quantity of
xanthones was significantly elevated in plants grown in vitro, in comparison to plants from
nature. Most of the obtained AtCKX transgenic centaury lines produced less secoiridoids
than plants from natural habitat or plants grown in vitro. On the contrary, it was determined
that the majority of the AtCKX transgenic centaury lines accumulated more xanthones than
plants grown in nature. As xanthones are increasingly being used for their pharmacological
properties, AtCKX transgenic centaury plants could be used as a useful source of plant
material for the production of novel drugs.
Keywords:
Centaurium erythraea Rafn.; Agrobacterium tumefaciens; qRT-PCR; Genetic transformation; AtCKX genes; CKX activity; Cytokinins; Secondary metabolites; Secoiridoids; XanthonesSource:
University of Belgrade, Faculty of Biology, 2012, 1-177URI
http://eteze.bg.ac.rs/application/showtheses?thesesId=14https://fedorabg.bg.ac.rs/fedora/get/o:2091/bdef:Content/download
http://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=
http://nardus.mpn.gov.rs/123456789/2021
https://radar.ibiss.bg.ac.rs/handle/123456789/2425
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IBISSAPA:
Trifunović, M. (2012). Genetička transformacija kičice (Centaurium erythraea Rafn.) AtCKX1 i AtCKX2 genima. University of Belgrade, Faculty of Biology. p. 177.Vancouver:
Trifunović M. Genetička transformacija kičice (Centaurium erythraea Rafn.) AtCKX1 i AtCKX2 genima [#dissertation]. Belgrade: University of Belgrade, Faculty of Biology; 2012. 177 p.Related items
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